Methods for making conjugates from disulfide-containing proteins

ABSTRACT

The invention provides methods to prepare protein conjugates from proteins having at least two cysteines. In one embodiment, a protein with a disulfide linkage is reduced to provide two free cysteines for reaction with a 1,3-dihaloacetone or similar reactant, linking the sulfur atoms of the two cysteines together. The ketone inserted between the sulfur atoms is then used to form a Schiff base to an aminated payload molecule, thus conjugating the protein to a payload. In another embodiment, two cysteine residues are tied together by reaction with a 1,3-dihaloacetone or similar reactant. The linkage between the sulfur atoms in each case holds the protein or peptide in a constrained conformation, while also providing a convenient place for attaching a payload with good specificity and efficiency.

BACKGROUND

A wide variety of chemical moieties (‘payloads’) have been covalentlyattached to enzymes, antibodies, and other large, polypeptides orproteins, to form conjugates. The payloads may be used to locate theprotein to which they are attached (e.g., labels), to modify thephysicochemical properties or stability of the protein (e.g.,PEGylation), to enable the protein to be attached to another molecule orprotein (coupling groups for connecting the conjugate to anothercompound or another conjugate), or to modify the function or activity ofthe payload or the protein (e.g., vaccine conjugates). The protein mayalso act as a carrier to deliver the attached payload to a particulartissue or cell type, such as in antibody-drug conjugates (ADCs). Classesof payloads that can be usefully linked to proteins include detectablemoieties (labels), anchoring moieties to attach the protein to a surfaceor another compound, antigens that elicit an immune response whenconjugated to a protein, coupling groups that react readily with achemically complementary coupling partner (thus connecting the proteinto another entity), and therapeutic moieties such as cytotoxins andother bioactive agents. Attaching these diverse structures to proteinsin a controlled and reproducible fashion is often critical for theconjugates to function correctly. Thus there is a need for a variety ofmethods to attach many types of payloads to many different proteins orpolypeptides.

A number of methods have been developed for attaching payloads toproteins to form protein conjugates. See, e.g., Sletten, E. M. andBertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6974-6998; Basle', E.;Joubert, N.; and Pucheault, M. Chemistry & Biology 2010, 17, 213-227. Insome protein conjugates, the method by which the protein and payload areconnected may have undetectable impact on the activity or relevantproperties of the conjugate; in other instances, the nature of thelinkage between protein and payload can significantly affect theactivity or properties of the conjugate. Sometimes it is critical tocontrol the number of payload moieties per protein, for example, or tocontrol the precise location where payloads are attached so they do notinterfere with functions of the protein. ADCs, for example, require theprotein to recognize and bind to surface structures on targeted cells toimpart selectivity, so a payload must not be positioned to interferewith binding of the antibody to the surface structures (antigen) thatthe antibody must recognize. See, e.g., Expert Opin. Biol. Ther. 2012,12, 1191-1206; Toxins 2011, 3, 848-883; and Laurent Ducry Drug Deliveryin Oncology: From Basic Research to Cancer Therapy, 1st Edition.Wiley-VCH Verlag GmbH & Co. KGaA. 2012, 12, 355-374.

Most methods for attaching payloads to proteins involve adding a linkingchemical structure (linker) between the protein and the particularpayload of interest. The linker provides a way to connect the payload ofinterest to the protein using available functional groups on eachmoiety. The linker often allows the distance between payload and proteinto be modulated, and may also include a cleavable portion that can belysed or degraded in vivo to release the payload from the protein whererelease is important for the payload to achieve its objectives. Forexample, in ADCs, it may be critical for the conjugate to break down andrelease the payload at a location where it can have a desired effect.Because of the diverse types of protein-payload conjugates that placedifferent demands on the manner in which the payload and protein areconnected, there is a continuing need for novel methods to link payloadsto proteins consistently and efficiently.

The most common methods for forming protein conjugates rely upon thechemical reactivity of certain amino acids that occur naturally in manynatural proteins: lysine and cysteine are often used because theyprovide a reactive site for connecting the payload to the protein.Lysine has a free amine group that can react with a suitableelectrophilic functionality on a linking group or payload, and cysteinecan react through its free sulfhydryl group. However, relying on thesenaturally occurring reactive sites can be complicated: when there aretoo many or too few of the particular type of amino acid in a protein ofinterest, for example, it becomes difficult to get just the right‘loading’ of payload on the protein. The high abundance of lysine onprotein surfaces makes site- and regio-selective conjugation difficult,and leads to heterogeneous products. In contrast, cysteines arecomparatively rare, and exist mainly in disulfide-linked pairs inproteins. Conjugation at cysteine often requires reduction of adisulfide, followed by reaction with a conjugation reagent (e.g.maleimide) to label individual cysteines separately. Because thisremoves a disulfide linkage, the protein structure and stability mightbe undermined by this process.

Proteins also often have more lysines than the optimum number ofpayloads to be attached: adding enough payload moieties to occupy all ofthe availably lysines in order to ensure a consistent, homogenousproduct may add too many payload molecules for optimum efficacy. Thiscan be avoided by using only some of the lysines for conjugation, butsuch partial or incomplete loading will generally provide aheterogeneous product, which can be problematic for a variety ofreasons—in the case of Mylotarg™, the first commercialized ADC, forexample, the heterogeneity of the ADC product seems likely to havecontributed to the issues that led to a decision to withdraw the productfrom registration. Fuenmayor, et al., Cancers, vol. 3, 3370-93 (2011).Also, even when enough amino acid groups of a particular type (e.g.,lysines) are present for optimal loading, some or all of them may be‘buried’ inside the protein when the protein is in its solutionconformation, rendering them effectively unavailable for conjugation, ormaking them ‘partially’ accessible which can also result inheterogeneity of the conjugate. Thus, while lysine can be a useful sitefor conjugation, in many situations it is not ideal.

The frequency of occurrence of cysteine in natural proteins is lowerthan that of lysine, and cysteine may be suitable for use as a site forconjugation where it is available in adequate numbers; where too fewcysteines are present, one or more may be inserted by standard proteinmodification methods. However, it is often preferable to avoid modifyingthe sequence of the natural protein by inserting a cysteine; besides,surface-accessible cysteines in natural proteins are often positionednear other cysteines to form disulfides, which may be important formaintaining the protein's active conformation. While it is not difficultto convert a disulfide into two free cysteines by reducing thedisulfide, doing so may disrupt the secondary or tertiary structure ofthe protein.

Some methods for attempting to insert a tether between cysteine residuesformed by reducing a disulfide on a protein have been reported. One suchmethod involves a sulfone-substituted methacrylate derivative.US2006/0210526. This method forms a reactive intermediate that requiresan elimination step before cyclization, and the conditions for thatmulti-step process can result in incomplete formation of a linker(tether) between cysteines, and the reaction conditions can even causeprotein denaturation. Another approach uses a maleimide derivative.WO2011/018613. However, the conjugate formed in this process suffersfrom stability problems because the Michael addition of the thiols onthe maleimide is reversible. There is thus a need for improved methodsto conjugate chemical moieties to proteins containing disulfide linkagesto form protein conjugates. In particular, methods are needed that usethe disulfide components (sulfhydryls) without giving up theconformation controlling effect of the disulfide, while also providingefficient conjugation, stability, and consistent payload/protein ratios.In addition, there is a need for stapling methods to hold proteins in aparticular conformation (see, e.g., Expert Opin. Drug Discov. (2011)6(9):937-963; Tetrahedron 55 (1999) 11711-11743) that also provide ameans to conjugate the stapled protein with a payload. The presentinvention provides such methods.

SUMMARY

In one aspect, the invention provides a method to use two cysteineresidues that form a disulfide on a protein's surface to link a payloadto the protein, forming a protein conjugate. The method involvesreducing the disulfide to provide two free thiol groups, and tying thetwo thiol groups together with a tether that keeps them in about thesame positions they occupied when they formed a disulfide. Keeping thecysteine groups in their same approximate positions minimizes anyadverse effect on the protein's conformation that may occur uponreduction of the disulfide. The tether that is introduced to link thetwo thiol groups together contains a reactive functional group that canbe used to attach a payload of interest. In some embodiments, the tethercontains a carbonyl group that is reactive enough to form an imine oroxime or hydrazone linkage with an external amine group, and the payloadis conjugated to the activated protein by forming such linkage. Forexample, the reduced protein can be reacted with a 1,3-dihalo acetonesuch as dichloroacetone or dibromoacetone, thereby inserting a 3-carbontether connecting the two sulfur atoms together. This may suitablysimulate the effect of the disulfide, i.e., to keep the protein in aconformation very similar to the one it had when the disulfide waspresent, while it also provides greater stability than the disulfide aswell as a place to attach a payload. The tethers used in the methods andcompositions of the invention provide a chemically reactive functionalgroup, and a protein containing this type of tether between two cysteinesulfur atoms is referred to herein as an activated protein. A payloadcan be attached to the activated protein using the functional group onthe tether. The tether formed by reacting the thiols of a protein with adihaloacetone provides a carbonyl as a site for conjugation, forexample. A payload containing a suitable amine (aminated payload),preferably an aminooxy or hydrazine, can easily be conjugated to such anactivated protein by forming a Schiff base between the aminefunctionality of the payload and the carbonyl group (ketone) of thetether. The process can use an unmodified payload molecule if itcontains a suitably reactive —NH₂ group, or a reactive amine such as—ONH₂ can be attached to the payload by conventional methods if one isneeded.

These methods can be applied to any protein having one or moreaccessible disulfide linkages, and are typically useful for naturalproteins having a molecular weight above 500 Da and typically above2,000 Da, where a disulfide is present in the native or active form ofthe protein. They can be used with proteins containing more than onedisulfide, such as 2-10, or typically up to 6 disulfide groups, at leastone of which is surface accessible sufficiently to be reduced byconventional disulfide reducing agents. These methods produce aconjugate containing at least one payload for each disulfide group thatis utilized, and the tether substantially retains the native or activeconformation of the protein.

In another embodiment, the invention provides a way to staple a proteinby tying two cysteine residues together, providing a rigidifiedconformation, wherein the stapling method ties two cysteines togetherwith a ketone-containing linkage that is then usable for conjugating thestapled protein to a payload. Stapling is accomplished by reacting aprotein containing at least two cysteine residues with a dihaloketonesuch as 1,3-dichloroacetone or 1,3-dibromoacetone to form a cyclizedprotein containing an [cys1]-S—CH₂—C(═O)—CH₂—S-[cys2] linkage, thenallowing the linkage to react with an aminooxy or hydrazino compound ofthe formula H₂N—X-L-PL to form a conjugate via Schiff base formation(including oximes and hydrazones) as further described herein. Theinvention includes methods of making these stapled conjugates as well asthe corresponding conjugated peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme illustrating an embodiment of the invention thatbegins with a disulfide-containing protein and provides aprotein-payload conjugate.

FIG. 2 illustrates coupling of two protein-payload conjugates havingcomplementary coupling groups as their payloads.

FIG. 3 shows mass spectra for the starting protein, activated proteinand protein conjugate of Example 1.

FIG. 4 shows the SDS Page gel dialysis of the azido substituted CRM197construct from Example 1.

FIG. 5 shows a schematic depiction of the activated protein for Example4 and LC-MS data for the activated (ketone-modified) protein.

FIG. 6 shows a schematic depiction of a protein-payload conjugatedescribed in Example 4 and LC-MS data for the product.

FIG. 7 shows a gel of the conjugate and reduced conjugate of Example 6,Method A, accompanied by a molecular weight ladder for comparison. Italso shows the LC-MS of the product of Method B, showing formation ofseveral different conjugates.

FIG. 8 shows LC-MS data for products of Step 1; Step 2, Method A (PL1);and Step 2, Method B (PL2) from Example 7.

FIG. 9 shows SDS PAGE gel and LC-MS data for the product of Example 8.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

‘Protein’ and polypeptide as used herein refer to peptides containingfive or more, typically ten or more amino acid residues connected byamide (peptide) bonds. Typically the proteins described herein comprisemainly or only naturally occurring amino acids, though the methods areequally useful with polypeptides that contain one or more non-naturalamino acids. Commonly (but not necessarily) the amino acids are mostlyor entirely of the L configuration and are selected from the common‘essential’ amino acids.

Abbreviations

DAR Drug to Antibody Ratio

DCM Dichloromethane

DIC Diisopropyl Carbodiimide

DIPEA Diisopropyl Ethyl Amine

EDT Ethane dithiol

HBTU N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate

MeCN acetonitrile

NMP N-methyl pyrrolidinone

PBS Phosphate-buffered saline

TCEP Tris(carboethoxyethyl)phosphine

TFA Trifluoroacetic acid

TIPS Triisopropyl silane

An example of one embodiment of the invention is shown in FIG. 1. TheFigure depicts a protein, represented as a shaded circle or sphere,having an exposed disulfide group. The disulfide is reduced, forming areduced protein having two free thiols derived from the disulfide. Thereduced protein is then allowed to react with a dihaloacetone or similarbis-electrophile (e.g., 1, 3-dichloroacetone or 1,3-dibromoacetone) toform an activated protein wherein the two thiols are linked togetherthrough a functionalized tether: the tether in this example contains afree carbonyl group that is relatively reactive toward Schiff baseformation. A payload molecule is then linked to the tether of theactivated protein via a Schiff base formation to provide a proteinconjugate. The payload in the example is attached via a linking group tothe tether. The compound of the formula H₂N—X-L-PL where PL is thepayload compound contains an activated amine group (H₂N—) that isconnected to PL by a linker L, and the amine is made especially reactiveby using an aminooxy or similar activated amine, —X—NH₂ where X is O orNH, which facilitates Schiff base formation between the ketone carbonyland the amine attached to PL. An alternative embodiment begins with aprotein having two free cysteine groups, such as the reduced protein inFIG. 1, and uses them to ‘staple’ the protein into a constrainedconformation while also providing an attachment point for a payload tobe conjugated onto the stapled protein.

The methods of the invention are suitable for use to form conjugatesfrom most proteins that contain at least one disulfide linkage betweentwo cysteines, or that contain two free cysteine residues that can beconnected together by reaction with a 1,3-dihaloacetone reactant.Typically, the protein is one where the two thiols react withdichloroacetone or dibromoacetone under conditions described herein toproduce at least 50% cross-linking of the two thiols, and frequently theextent of cross-linking is at least about 70%, 80% or 90%.

The two cysteines to be linked together may be on a single polypeptide,or they may be on separate polypeptides that form a protein complex. Incertain embodiments, the methods utilize a protein having 1-6 disulfidelinkages, or 2-6 free cysteine residues, and involve reduction of atleast one of these disulfides. The disulfide-containing protein can beany polypeptide having at least 5 amino acid residues, preferably atleast 10 amino acids, that contains a disulfide linkage within a singlepolypeptide sequence, or a protein complex where a disulfide connectsone polypeptide sequence to another amino acid or polypeptide, providedthe complex does not dissociate rapidly when the disulfide is reducedfor insertion of the tether between the sulfur atoms. Typical proteinsfor use in the methods of the invention include cyclic peptides andlinear peptides containing about 5 to about 5000 amino acids, typicallyat least 10 amino acids and up to about 1000, including functionalproteins such as enzymes or receptors; protein complexes having at leastone disulfide linkage (often connecting two separate polypeptidestrands); structural proteins; proteins used as vaccine scaffolds suchas CRM197 or other proteins having adjuvant activity; and antibodies orantibody fragments. Particularly useful proteins for these methodsinclude antibodies, especially monoclonal antibodies includingengineered antibodies, modified antibodies and antibody fragments;vaccine carrier proteins such as CRM197; and single-stranded proteinshaving at least one disulfide linkage or at least two cysteine residuesand having a molecular weight between 500 and 500,000, typically between1,000 and 200,000. Methods for engineering an antibody or other proteinto introduce one or more cysteine residues, for example, and formodifying antibodies are well known in the art.

The methods are especially useful with antibodies and antibodyfragments, including IgG, which have up to 4 accessible disulfide bonds.The methods are also especially useful with vaccine carrier proteinssuch as diphtheria toxoid, non-toxic cross-reactive material ofdiphtheria toxin (197) (CRM197), tetanus toxoid, keyhole limpethemocyanin, N. meningitidis outer membrane protein, and non-typeable H.influenza-derived protein D. These vaccine carrier proteins can befunctionalized with antigens by known methods and/or by the methodsdisclosed herein. The present methods can also be used to attach anadjuvant compound such as a TLR agonist (a ligand of TLR3, TLR4, TLR5,TLR7, TLR8, or TLR9) including imiquimod, imidazoquinolines, andgardiquimod, PRR ligands, RLR ligands, NOD2 ligands, cyclic di-AMP,cyclic di-GMP, flagellin, monophosphoryl lipid A, N-glycolatedmuramuldipeptide, CpG oligodeoxynucleotides (CpG ODN), triacylatedlipoprotein, or poly (I:C), to provide an enhanced immune response.

The disulfide linkages of disulfide-containing proteins for use in themethods and compositions of the invention are reduced to form two freethiol groups: methods for such reduction are well known in the art. Insome embodiments, the reduction is performed using a reducing agent thatselectively reduces disulfide linkages that are readily accessible tosolvent around the protein: one suitable reducing agent istris(2-carboethoxy)phosphine (TCEP) and its salts—see AnalyticalBiochemistry 273, 73-80 (1999). Other known disulfide-reducing agentssuch as dithiothreitol, 2-mercaptoethanol, cysteamine, anddithiobutylamine (J M Perkel, Chem. Eng'g News, Feb. 29, 2012; Lukesh,et al., J. Am. Chem. Soc., 134, 4057-59 (2012)) and trialkyl phosphinessuch as tributyl phosphine (WO2008/157380) can also be used. Methods forreducing disulfides in proteins are well known in the art.

The group ‘X’ connecting the nitrogen that forms a Schiff base with thecarbonyl of the tethering linkage to the linking group-payload portionof the added moiety (-L-PL) can be oxygen, or it can be optionallysubstituted nitrogen. When X is oxygen (O), the linkage comprises anoxime, which is typically stable in vivo. The linking group L connects Xto at least one and optionally two or more payload groups; for example,the portion -L-PL could be —CH(CH₂O-PL)₂ so that a single modifieddisulfide links the protein to two payload molecules, which can be thesame or different. When X is nitrogen, it can be —NH or it can be —NR,where R is C₁₋₄ alkyl (e.g., methyl, ethyl, propyl, butyl);alternatively, the nitrogen can be —N-L-PL, thus carrying twolinker-payload moieties. In the latter embodiments, the two linkers canbe the same or different, and the two payloads can be the same ordifferent. In some embodiments both linkers and payloads are the same,so the result is a conjugate where a single tethering linkage carriestwo identical payloads:

In other embodiments, the two linkers and two payloads are different,and PL¹ and PL² may even belong to different payload classes.

The linking group L can be any suitable organic linkage that connectsthe payload compound to —X—NH₂. Some examples of suitable linkagesinclude [X]—(CH₂)₁₋₆—[PL]; [X]—CH₂C(═O)—[PL]; [X]—CH₂C(═O)—NH—[PL];[X]—CH₂C(═O)—O—[PL]; [X]—(CH₂CH₂O)n-[PL]; [X]-Phenyl-C(O)NH—[PL],[X]—(CH₂)₁₋₁₀—C(═O)—NH—(CH₂)₂₋₁₀—NH—C(═O)—(CH₂)₀₋₁₀—(OCH₂CH₂)₀₋₁₀-(AA)₀₋₁₀-[PL](AA can be any of the essential amino acids, e.g. glu, gly, ala, asp,etc.), and the like, where n is typically 1-20, and [X] and [PL]respectively indicate which end of the linker is attached to X and whichto PL. In some embodiments, the linker L can have two or three payloadsattached to increase payload loading on the conjugate, and where morethan one payload is attached to a given linker the payloads can be thesame or different. Suitable linkers also include combinations of thecomponents of these groups: the nature of the linker is not important tothe practice of the invention and can be based on convenience andavailability of methods for attachment to at least one payload PL, or ondesired physicochemical properties for the conjugate. Selection of asuitable linker is within the level of ordinary skill and depends on thestructure of the Payload and available methods for modifying it toattach linker L. Typically the linker is attached at one or both endsvia an amide or ester group; frequently the linker L contains a peptidebond or ester to allow in vivo lysis by protease or esterase activities(for example val-cit, a dipeptide that is cleaved by cathepsin B, orGly-phe-leu-gly, which is also cleavable by cathepsin B); optionally itcontains one or more ethylene oxide units (—OCH₂CH₂—); and in manyembodiments it contains at least one and up to six amino acid moieties.Suitable embodiments of L may also comprise one or more spacers, whichmay be selected from the following groups:

-   -   (a) a bond, —O—, —S—, —S—S—, —NH—, —N((C₁-C₆)alkyl)-,        —NH—C(O)—NH—, —C(O)—NH—, —NH—C(O)—;    -   (b) (C₁-C₂₀)alkylene, (C₂-C₂₀)alkenylene, (C₂-C₂₀)alkynylene,        —Z—(C₁-C₂₀)alkylene-, —Z—(C₂-C₂₀)alkenylene,        —Z—(C₂-C₂₀)alkynylene, (C₁-C₂₀)alkylene-Z—(C₁-C₂₀)alkylene,        (C₂-C₂₀)alkenylene-Z—(C₂-C₂₀)alkenylene,        (C₂-C₂₀)alkynylene-Z—(C₂-C₂₀)alkynylene, where Z is —NH—,        —N(C₁-C₆)alkyl)-, —NH—C(O)—NH—, —C(O)—NH—, —NH—C(O)—,        (C₃-C₇)cycloalkylene, phenylene, heteroarylene, or heterocyclene        and where said (C₁-C₂₀)alkylene, said (C₂-C₂₀)alkenylene, and        said (C₂-C₂₀)alkynylene moieties each independently optionally        contain one or more oxygen atoms interdispersed within said        moieties, such that the oxygen atoms are separated by at least        one and preferably two carbon atoms;    -   (c) (C₃-C₇)cycloalkylene,        (C₃-C₇)cycloalkylene-Y—(C₃-C₇)cycloalkylene,        —Y—(C₃-C₇)cycloalkylene, phenylene, —Y-phenylene,        phenylene-Y-phenylene, heteroarylene, Y-heteroarylene,        heteroarylene-Y-heteroarylene, heterocyclene, —Y-heterocyclene,        or heterocyclene-Y-heterocyclene, where Y is (C₁-C₂₀)alkylene,        (C₂-C₂₀)alkenylene, (C₂-C₂₀)alkynylene, —O—, —C(O)—, —S—, —NH—,        —N((C₁-C₆)alkyl)-, —NH—C(O)—NH—, —C(O)—NH—, or —NH—C(O)— and        where said (C₃-C₇)cycloalkylene, said phenylene, said        heteroarylene, and said heterocyclene moieties are each        individually optionally substituted with 1 to 3 substituents        selected from halo, (C₁-C₄)alkyl or        halo-substituted(C₁-C₄)alkyl;    -   (d) —[OCH₂CH₂]_(v)—, where v is 1-2,000, preferably 1-10; and    -   (e) a peptide comprising 1 to 100 amino acids, preferably 1-30        or 1-6 amino acids.

Furthermore, L can be or can comprise a cleavable linker such as Val-Cit(valine-citrulline, a dipeptide that is selectively cleaved by cathepsinB) or val-cit-PABC (valine-citrulline p-aminobenzylcarbamate, seeBioconjugate Chem. 19(10), 1960-63 (2008)), a disulfide, or a linkercleaved by glucuronidase, such as the linker present in this formula:

-   -   where Protein represents a protein for conjugation, X is O or NR        as described above, PL represents a Payload as described herein,        and L¹ and L² are independently optional linkers such as the        groups L described above. (ACS Med. Chem. Letters, vol. 1,        277-280 (2010).

The Payload (PL) can be any moiety that is useful to attach to aprotein. Many examples of compounds that can be usefully attached toproteins are known in the art. Examples include label moieties thatenable a user to locate or identify the protein, including chelatorsthat bind metal ions to provide detectability of the conjugate; bindingmoieties such as biotin or avidin, polynucleotides, antibodies orfragments thereof, poly-Arg or poly-lys containing 5-15 amino acidresidues, etc., that make it easy to purify or isolate the protein oraffix it to a surface; property-modifying groups such as fatty acidgroups or polyethylene glycol (PEG); antigenic groups such aspolysaccharides or cell surface proteins that are characteristic of aparticular type of cell or bacterium; coupling groups that enable themodified protein or peptide to be attached to another molecule to makemore complex conjugates, such as bispecific antibodies (see FIG. 2); andbioactive compounds including nucleic acids like RNA, DNA, mRNA, siRNA,and fragments of these; pharmaceutical compounds such as varioustherapeutic drugs; and radionuclides and cytotoxins, which can hitchhikeon the protein to a desired tissue or cell where they can produce adesired effect. These hitchhiking compounds may act while they remainconjugated to the protein or a portion thereof, or they may first detachfrom the protein if the linking group is one that can readily cleave invivo. Suitable pharmaceutical payloads for use with these methodsinclude microtubule inhibitors, topoisomerase I inhibitors,intercalating agents, inhibitors of intracellular signaling pathways,kinase inhibitors, transcription inhibitors such as siRNAs, aRNAs, andmiRNAs, and DNA minor groove binders; these payloads include compoundclasses such as maytansinoids, auristatins, amanitins, calicheamycins,psymberins, duocarmycins, anthracyclins, camptothecins, doxorubicins,taxols, pyrrolobenzodiazepines, and the like.

Specific examples of these pharmaceutical payloads having therapeutic ordiagnostic uses include paclitaxel, docetaxel, etoposide, tenoposide,vincristine, vinblastine, colchicine, doxorubicin, daunorubicin,mithramycin, actinomycin, glucorticoids, puromycin, epirubicin,cyclophosphamide, methotrexate, cytarabine, f-fluorouracil, platins,streptozotocin, minomycin C, anthracyclines, dactinomycin oractinomycin, bleomycin, mithramycin, anthramycin, duocarmycins,ifosfamide, mitoxantrone, daunomycin, carminomycin, animoterin,melphalan, esperamicins, lexitropsins, auristatins (e.g., auristatin E,auristatin F, AEB, AEVB, AEFP, MMAE, MMAF), eleuthorobin, netropsin,podophyllotoxins, maytansiods including maytansine and DM1, andcombretestatins.

Suitable coupling groups that can be used as payloads (groups that canbe used to couple the conjugate to another moiety) include maleimide,thiols, alpha-halo ketones (e.g., —C(═O)—CH₂—X where X is chloro, bromoor iodo), carboxylic acids, amines, hydroxyls, alkenes, alkynesincluding cyclic octynes that can be used in copper-free ‘click’chemistry, azide, and the like. Methods to use these coupling groups toconnect the conjugates of the invention to other compounds havingcomplementary coupling groups are well known in the art, and includeMichael addition of a thiol to a maleimide, alkylation of a thiol withan alpha-haloketone, amide bond formation between amine and a carboxylicacid, ‘click’ chemistry (see, e.g., Meldal, et al., Chem Rev., vol 108,2952-3015 (2008)) to link an azide to an alkyne by forming a1,2,3-triazole ring, and ‘copper-free click’ chemistry. See e.g.,Meeuwissen, et al. Polymer Chemistry, vol. 3, 1783-95 (2012).‘Complementary’ coupling groups are two coupling groups that readilycombine to form a covalent bond, such as the pairs mentioned above(carboxylate plus amine to form an amide; azide plus alkyne to form a1,2,3-triazole; maleimide plus thiol, where the thiol adds to the doublebond via a Michael addition; alpha-halo ketone plus thiol which form analpha-thio ketone by alkylation of the thiol; etc.) A depiction of aconjugate containing a coupling group as payload being coupled with asecond conjugate containing a complementary coupling group is providedin FIG. 2. In particular examples, a coupling group to serve as aPayload (PL) is selected from the group consisting of halogen, —C≡CH,—C═CH₂, —OH, —SH, —SO₂—CH═CH₂, —O—NH₂, —N₃, —O—P(O)(OH)₂, —C(O)—H,—C(O)—CH₃, —NH—C(O)—CH₂—I, maleimidyl, 3,5-dioxo-1,2,4-triazolidin-4-yl,1H-pyrrole-2,5-dione-1-yl, pyridin-2-yl-disulfanyl,tetrahydro-1H-thieno[3,4-d]imidazol-2(3H)-one-4-yl,1-carbonyloxy-2,5-dioxopyrrolidine, sodium1-carbonyloxy-2,5-dioxopyrrolidine-3-sulfonate, —SSR¹, —C(O)—OR¹,—N(R¹)H, —NH—N(R¹)H, where R¹ is H or (C₁-C₆)alkyl, and —C(O)—R², whereR² is H, (C₁-C₄)alkyl, halo-substituted(C₁-C₄)alkyl, —CH═CH₂, N(R¹)H, or—NH—N(R¹)H. When these Payloads are used as an initial payload (PL^(a)),the conjugate can be reacted with a compound comprising a second payload(PL^(b)), and may introduce an additional linker L′ in forming a newconjugate:

Note that in these reactions, the person of skill in the art willunderstand that the L and L′ may retain a portion of PL^(a) and/or R,depending upon the reaction being used to connect the new Payload,PL^(b).

The following enumerated embodiments illustrate particular aspects ofthe invention.

-   -   1. A method to form a Protein-Payload conjugate from a protein        that comprises at least two cysteine residues, where the method        comprises:        -   a) contacting the protein with a functionalized tethering            compound under conditions where the functionalized tethering            compound reacts with two free thiol groups on the protein to            form an activated protein having the two thiol groups            covalently linked together by a functionalized tether; and        -   b) if the functionalized tether is not already linked to a            payload, contacting the activated protein with a            functionalized payload compound to form a covalent            attachment of the payload with the functionalized tether to            form a Protein-Payload conjugate.

In some embodiments, the protein has two cysteines forming a disulfidebond, and the disulfide is reduced before contacting the protein withthe functionalized tethering compound. In other embodiments, the proteinhas two free cysteines (i.e., cysteines having free —SH groups) that areable to get near enough to each other to be tied together by thefunctionalized tethering compound. In each case, the functionalizedtethering compound reacts with two cysteine residues, tying themtogether and providing a constraint on the protein's conformation. Whenthe two cysteines come from a disulfide, the constraint provided by thetether between the sulfur atoms imitates to a useful degree theconstraint that would have been lost by reduction of the disulfide,which is often important in maintaining the protein in a desired orbioactive conformation.

The functionalized tethering compound is a compound having two reactivegroups that readily react with a thiol (the —SH of a cysteine) underconditions where the protein is stable and soluble, typically in anaqueous buffered medium at a temperature between about 00 and 50° C., toform covalent bonds between the sulfhydryls of the cysteines and theframework of the tethering compound. The functionalized tetheringcompound also has either an additional functional group like a carbonylthat can be used to conjugate with another moiety that includes apayload, or it is already attached to a payload. In preferredembodiments, the functionalized tethering compound is a1,3-dihaloacetone, typically 1,3-dichloro- or 1,3-dibromo-acetone.

-   -   2. The method of embodiment 1, wherein the protein has two        cysteine residues joined together by a disulfide linkage before        it is contacted with the functionalized tethering agent, and        wherein the disulfide is reduced to provide a protein with two        free cysteine residues for use in step a) of embodiment 1.        Methods for reducing the disulfide are well known in the art,        and are described herein. In certain embodiments, the disulfide        is reduced with a selective reducing agent like TCEP, which can        selectively reduce solvent-exposed disulfides without reducing        buried ones that help maintain the protein in a particular        conformation.    -   3. The method of embodiment 1 or 2, wherein the functionalized        tethering compound is a dihaloacetone derivative, and the        functionalized tether linking the sulfur atoms together in the        Protein-Payload conjugate is-CHR—C(═Z)—CHR—, where Z is O or        NR′, and each R is independently H, phenyl, C1-C4 alkoxy, or        C1-C4 alkyl, and R′ represents a linking group attached to a        payload. R′ can thus represent a group of the formula -L-PL:        suitable linking groups (L) and payloads (PL) for R′ are        described herein. In certain embodiments, the functionalized        tethering compound is 1,3-dichloroacetone or 1,3-dibromoacetone,        and Z is O.    -   4. The method of any of embodiments 1-3, wherein the        functionalized payload is a compound of the formula H₂N—O-L-PL        or H₂N—NR′-L-PL, where L represents a linking group; and PL        represents at least one payload molecule.    -   5. The method of any of embodiments 1-3, wherein the        functionalized payload is a compound of the formula H₂N—O-L-PL.    -   6. The method of embodiment 1, wherein the activated Protein        comprises at least one ketone having the formula

-   -   where the circle represents the protein, and each sulfur atom is        from the sulfhydryl of a cysteine residue of the protein.    -   7. The method of any of the preceding embodiments, wherein the        Protein-Payload conjugate comprises a group of the formula:

-   -   wherein X is O or NH, L represents a Linker, and PL represents        at least one Payload group.    -   8. The method of any of the preceding embodiments, wherein the        Protein is an antibody. The antibody can be a polyclonal or        monoclonal antibody or an antibody fragment, and can be modified        by methods known in the art such as PEGylation. In preferred        embodiments, the antibody is a monoclonal antibody and may be        engineered to modify one or more residues while retaining the        bioactivity associated with the antibody; methods for        engineering antibodies are well known in the art.    -   9. The method of any of embodiments 1-7, wherein the Protein is        a vaccine carrier.    -   10. The method of any of embodiments 1-8, wherein the Payload        comprises a therapeutic agent.    -   11. The method of any of embodiments 1-8, wherein the Payload        comprises a detectable label or a binding group. Suitable        binding groups include cell surface markers (e.g.,        polysaccharides or proteins) that would allow the conjugate to        bind to a particular cell type, as well as binding groups like        poly-nucleotides and polypeptides known to be useful for binding        a protein or conjugate to a surface, cell, or subcellular        organelle.    -   12. The method of embodiment 9, wherein the Payload comprises an        antigen. Typically, these would be bacterial or viral antigens,        attached to the conjugate to provoke an immune response.    -   13. The method of embodiment 8, wherein L comprises a cleavable        linking moiety. Val-Cit, Val-Cit-PABC, Gly-phe-leu-gly, and        glucuronidase-cleavable groups such as those described herein        are suitable.    -   14. The method of any of embodiments 1-13, wherein L comprises        at least one amino acid. In some embodiments, two or more amino        acids may be included.    -   15. The method of any one of embodiments 1-14, wherein L        comprises at least one spacer selected from:    -   (a) a bond, —O—, —S—, —NH—, —N((C₂-C₆)alkyl)H—, —NH—C(O)—NH—,        —C(O)—NH—, —NH—C(O)—;    -   (b) (C₁-C₂₀)alkylene, (C₂-C₂₀)alkenylene, (C₂-C₂₀)alkynylene,        —Z—(C₁-C₂₀)alkylene-, —Z—(C₂-C₂₀)alkenylene,        —Z—(C₂-C₂₀)alkynylene, (C₁-C₂₀)alkylene-Z—(C₁-C₂₀)alkylene,        (C₂-C₂₀)alkenylene-Z—(C₂-C₂₀)alkenylene,        (C₂-C₂₀)alkynylene-Z—(C₂-C₂₀)alkynylene, where Z is —NH—,        —N(C₂-C₆)alkyl)H—, —NH—C(O)—NH—, —C(O)—NH—, —NH—C(O)—,        (C₃-C₇)cycloalkylene, phenylene, heteroarylene, or heterocyclene        and where said (C₁-C₂₀)alkylene, said (C₂-C₂₀)alkenylene, and        said (C₂-C₂₀)alkynylene moieties each independently optionally        contain 1-10 oxygen atoms interdispersed within said moieties;    -   (c) (C₃-C₇)cycloalkylene,        (C₃-C₇)cycloalkylene-Y—(C₃-C₇)cycloalkylene,        —Y—(C₃—C)cycloalkylene, phenylene, —Y-phenylene,        phenylene-Y-phenylene, heteroarylene, Y-heteroarylene,        heteroarylene-Y-heteroarylene, heterocyclene, —Y-heterocyclene,        or heterocyclene-Y-heterocyclene, where Y is (C₁-C₂₀)alkylene,        (C₂-C₂₀)alkenylene, (C₂-C₂₀)alkynylene, —O—, —C(O)—, —S—, —NH—,        —N((C₁-C₆)alkyl)H—, —NH—C(O)—NH—, —C(O)—NH—, or —NH—C(O)— and        where said (C₃—C)cycloalkylene, said phenylene, said        heteroarylene, and said heterocyclene moieties are each        individually optionally substituted with 1 to 3 substituents        selected from halo, (C₁-C₄)alkyl or        halo-substituted(C₁-C₄)alkyl;    -   (d) —[OCH₂CH₂]_(v)—, where v is 1-2,000; and    -   (e) a peptide comprising 1 to 100 amino acids.    -   16. A protein-payload conjugate of the formula:

-   -   -   wherein the circle represents a protein having at least 5            amino acids;        -   X is NH, N(C₁₋₄alkyl), N-L²-PL², or O;        -   L and L² are each a linking group and can be the same or            different; and        -   PL and PL² are payload moieties that can be the same or            different.

    -   17. The protein-payload conjugate of embodiment 16, wherein X is        O.

    -   18. The protein-payload conjugate of embodiment 16 or 17,        wherein the protein is an antibody.

    -   19. The protein-payload conjugate of embodiment 16 or 17,        wherein the protein is a vaccine carrier.

    -   20. The protein-payload conjugate of any of embodiments 16-18,        wherein the payload is a therapeutic agent, a detectable label,        an antigen, or a binding group.

    -   21. The protein-payload conjugate of any of embodiments 16-20,        wherein L comprises a cleavable linker.

    -   22. The protein-payload conjugate of any of embodiments 16-21,        wherein L comprises at least one amino acid.

    -   23. The protein-payload conjugate of any one of embodiments        16-22, wherein L comprises at least one spacer of the formula        (CH₂)₁₋₆ or (CH₂CH₂O)₁₄.

    -   24: A method to stabilize a protein that contains a disulfide        bond or constrain it in a desired conformation, comprising:

    -   (a) reducing the disulfide bond to form two free thiol groups,        and

    -   (b) introducing a —CH₂—C(═N—X-L-PL)-CH₂— linkage that connects        the two thiol groups together, wherein X is NH, N(C₁₋₄ alkyl),        N-L²-PL², or O;        -   L and L² are each a linking group and can be the same or            different; and        -   PL and PL² are payload moieties that can be the same or            different.

    -   25. The method of embodiment 24, wherein the linkage        —CH₂—C(═O)—CH₂— is used to connect the two free thiols together,        followed by reaction of the carbonyl of the linkage with a group        of the formula H₂N—X-L-PL.

    -   26: A method to staple a protein that contains two or more        cysteine residues to form a cyclized protein conjugate,        comprising:

    -   (a) introducing a —CH₂—C(═O)—CH₂— linkage that connects the        thiol groups of two cysteine residues together to cyclize a        portion of the protein, and

    -   (b) contacting the cyclized protein with an aminated payload of        formula H₂N—X-L-PL,        -   wherein X is NH, N(C₁ alkyl), N-L²-PL², or O;        -   L and L² are each a linking group and can be the same or            different; and        -   PL and PL² are payload moieties that can be the same or            different;            to form a stapled conjugate of the cyclized protein.

    -   27. The method of embodiment 26, wherein 1,3-dihaloacetone is        used to connect two cysteine thiols together with the        —CH₂—C(═O)—CH₂— linkage, followed by reaction of the carbonyl of        the linkage with an aminated payload compound of the formula        H₂N—O-L-PL.

The methods of the invention, as summarized in FIG. 1, involve reducinga disulfide of a protein to be modified, forming a reduced protein thatcontains two free thiol groups. The reduced protein is contacted with afunctionalized tethering compound that is capable of reacting with bothof the free thiols on the reduced protein to tether the free thiolstogether, while also retaining at least one functional group on thetether that is suitable for attaching a payload. In some embodiments,the functional group on the tether is a carbonyl group, e.g., the ketoneobtained when the free thiols are allowed to react with a1,3-dihaloketone. Because the free thiols are strongly nucleophilic,they react readily with electrophiles such as alkyl halides or alkyltosylates, via irreversible reactions that involve displacing a leavinggroup and forming a covalent sulfur-carbon bond. Some suitable examplesof functionalized carbonyl-containing tethering compounds include1,3-dichloroacetone and 1,3-dibromoacetone. These reagents have beenused to provide stabilization of disulfide moieties in small cyclicpeptides by tethering sulfhydryls together. See e.g. WO2008/157380(reaction of dichloroacetone with a reduced cyclic pentapeptide,followed by reduction of the carbonyl). Sulfonates of1,3-dihydroxyacetone (e.g., mesylate, triflate, phenylsulfonate,tosylate, and the like) can also be used. These reagents aresufficiently reactive toward the free thiols of a reduced protein toprovide reasonably rapid reaction to form an activated protein with twocysteine residues tethered together, wherein each of the free thiols iscovalently attached to the functionalized tethering group.

The reduced protein and functionalized tethering compound are contactedunder conditions suitable to promote reaction between the tetheringcompound and the two free thiols of the reduced protein, andparticularly under conditions of concentration and temperature thatfavor causing both of the free thiols that were previously joined in adisulfide bond to react with a single molecule of the tethering compoundso they are once again tied together, but now with a short tetherconnecting them instead of a direct disulfide bond. This reaction formsan activated protein as illustrated in FIG. 1, having a functionalizedtether [—CH₂C(O)—CH₂—] between the two sulfur atoms. The tether in FIG.1 includes a carbonyl that can be used to efficiently attach a payloadvia clean and efficient Schiff base formation chemistry.

It is understood throughout this discussion that the protein, eventhough it is depicted as a circle or sphere, can be a small polypeptideof fewer than 10 amino acids or a large enzyme or complex of two or moresubunits or distinct proteins. The two sulfur atoms of the disulfide canbe on one subunit of a multimeric complex, or they can be on differentsubunits. In addition to the disulfide participating in thetransformations described herein, the protein may also contain otherdisulfide linkages that may be reduced and functionalized, or may not bereduced due to their location within the protein. While only a singledisulfide, tethering group, or conjugation is shown, it is understoodthat a polypeptide or protein comprising one such disulfide, tetheringgroup or conjugation may also contain more than one. The methods of theinvention can utilize known methods to selectively reducesolvent-accessible disulfide linkages near the surface of the foldedprotein, often without reducing ‘buried’ disulfides that may beessential for maintaining the overall shape and functionality of theprotein, or for keeping two subunits linked together in a multi-subunitcomplex. As the examples illustrate, a protein or polypeptide cancontain more than one functionalized tethering group, and thus cancontain more than one conjugation site, even though only one istypically depicted for simplicity.

Once the activated protein has been formed, a payload can be attached tothe functionalized tether. For example, an amine-containing (oraminated) payload can be attached to the tether formed fromdihaloacetone by forming a Schiff base between the payload's amine andthe tether's ketone. Suitable payload compounds contain an —NH₂ aminegroup that is accessible and reactive; in preferred embodiments, theamine is one that is activated toward forming a Schiff base. Examples ofsuitable amines include oxyamines (X=O), thioamines (X=S), andhydrazines (X=NH), for example: these heteroatom-substituted amines areknown to condense readily with ketones such as the one on the tether ofan activated protein formed from a dihaloacetone as shown in FIG. 1.

The activated protein is typically contacted with an amino-containingpayload without purification or isolation of the activated protein. Afree —NH₂ group on the payload (PL) can be used if available, but ifnone is available, one can be added via a linking group as illustratedin FIG. 1 and in the examples. In some embodiments, once the activatedprotein is generated, the amino-payload is added to the reaction mixturewhere the activated protein was formed under conditions that promoteformation of the desired Schiff base. The amino-payload then reacts viaits amine group with the carbonyl of the activated protein asillustrated in FIG. 1, thereby forming the desired Protein-Payloadconjugate, wherein X is O, NH or substituted N; L is a linking group;and PL represents a payload.

EXAMPLES

The Following HPLC Methods are Used in the Examples Below.

Method A:

Eluent A: water+0.1% Formic acid, Eluent B: Acetonitrile+0.08% Formicacid

Gradient: from 3 to 80% B in 2 min—Flow 1.0 ml/min. Column: ProswiftMonolith 4.6*50 mm 40° C.

Method B:

Eluent A: water+0.1% Formic acid, Eluent B: Acetonitrile+0.04% Formicacid

Gradient: from 3 to 80% B in 2 min—Flow 1.0 ml/min. Column: ProswiftMonolith 4.6*50 mm 40° C.

Method C:

Eluent A: water+3.75 mM ammonium acetate+2% acetonitrile, Eluent B:Acetonitrile

Gradient: from 2 to 98% B in 1.7 min—Flow 1.0 ml/min. Column: AcquityCSH 2.1*50 mm 50° C.

Method D (HRMS):

Eluent A: water+0.05% Formic acid+3.75 mM ammonium acetate, Eluent B:

Acetonitrile+0.04% Formic acid.

Gradient: from 2 to 98% B in 4.4 min—Flow 1.0 ml/min. Column: AcquityCSH 2.1*50 mm 50° C.

Synthesis of a Linker

2-Chlorotrityl chloride resin (1.55 mmol/g) (0.500 g, 0.775 mmol) in 100mL glassware was swollen in DCM (20 ml) for 30 min and it was drained. Asuspension of 2-(aminooxy)acetic acid hemihydrochloride (0.338 g, 3.10mmol) and DIPEA (1.354 ml, 7.75 mmol) in NMP (7 ml)/DCM (4 ml) was addedto the resin, which was shaken for 5 h. Solvent was drained. Resin wasrinsed with DCM/MeOH/DIPEA (17/2/1, 40 mL), DCM (50 mL), NMP (50 mL) andDCM (50 mL) respectively. Resulting resin was dried with KOH/NaOHovernight.

Resin (0.775 mmol) in 100 mL glassware was swollen in DCM (20 ml) for 30min and it was drained. Into a suspension of (9H-fluoren-9-yl)methyl2-aminoethylcarbamate hydrochloride (0.081 g, 0.775 mmol), HOAt (0.422g, 3.10 mmol) and DIPEA (1.354 ml, 7.75 mmol) in NMP (8 ml) was addedHBTU (1.176 g, 3.10 mmol) in NMP (2.5 ml), which was shaken for 2 h atRT. The solvent was drained, and resin was rinsed with NMP (10 mL) andDCM (10 mL) sequentially. The resulting resin was dried overnight.

Resin (0.775 mmol) was charged into a reaction vessel. 10 mL of 20%PIPERIDINE/NMP (v/v) was added, and the suspension was agitated at roomtemperature for 5 min. After solvent was drained, additional 10 mL of20% PIPERIDINE/NMP (v/v) was added and agitated for 20 min at roomtemperature. A solution of HOAt (0.316 g, 2.325 mmol) and1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecan-12-oic acid (0.896g, 2.325 mmol) in NMP (8 mL) was added into resin and DIC (0.362 ml,2.325 mmol) in NMP (1 ml) was added. The reaction mixture was agitatedfor 2 h at room temperature, and the resin was filtered off and rinsedwith NMP (10 ml) four times. The resulting resin was dried overnight.

Resin (0.775 mmol) was charged into a reaction vessel. 10 mL of 20%PIPERIDINE/NMP (v/v) was added into resin, and the suspension wasagitated at room temperature for 5 min. After solvent was drained,additional 10 mL of 20% PIPERIDINE/NMP (v/v) was added and agitated for20 min at room temperature. A solution of HOAt (0.316 g, 2.325 mmol) andFmoc-Glu-OtBu (0.989 g, 2.325 mmol) in NMP (8 ml) was added into resinand DIC (0.362 ml, 2.325 mmol) in NMP (2.00 ml) was added. The reactionmixture was agitated for 2 h at room temperature. Resin was filtered offand rinsed with NMP (10 ml) four times. The resulting resin was driedovernight.

Attaching a Payload to Linker

Resin (2-chlorotrityl chloride resin, 0.775 mmol) was charged into areaction vessel. 10 mL of 20% PIPERIDINE/NMP (0.775 mmol, v/v) was addedinto resin, and the suspension was agitated at room temperature for 5min. After solvent was drained, additional 10 mL of 20% PIPERIDINE/NMP(0.775 mmol) was added and agitated for 20 min at room temperature. Asolution of 18-tert-butoxy-18-oxooctadecanoic acid (0.862 g, 2.325 mmol)and HOAt (0.316 g, 2.325 mmol) in NMP (8 mL) was added into resin andDIC (0.362 mL, 2.325 mmol) in NMP (2.00 ml) was added. The reactionmixture was agitated for 4 h at room temperature. Resin was filtered offand rinsed with NMP (10 ml) four times. The resulting resin was driedovernight,

Resin from the preceding step (0.775 mmol) was treated with 20 mL ofcleavage cocktail (TFA/TIPS/water=95/2.5/2.5, v/v) for 1.5 h at roomtemperature. Resin was removed by filtration and rinsed with TFA. Thefiltrate was concentrated in vacuo. RP-HPLC with C18 column eluting with15-50% MeCN/water plus 0.1% TFA gave(S)-1-(aminooxy)-19-carboxy-2,7,16,21-tetraoxo-9,12-dioxa-3,6,15,20-tetraazaoctatriacontan-38-oicacid with 2,2,2-trifluoroacetic acid (1:1) (207 mg, 0.294 mmol, 37.9%yield) (PL1). HRMS[M+1] (method D); 704.4459 (observed), 704.4486(expected).

Example 1

CRM197 (ref: G. Giannini and R. Rappuoli, Nucleic Acids Res., 1984, 25,4063.) was treated with TCEP (xx), resulting in reduction of theC201-C185 disulfide, with little or no reduction of the C451-C471disulfide (see Example 3). The reduced CRM197 was treated with1,3-dichloroacetone to provide the activated protein, having theC451-C471 disulfide intact and with C201 tethered to C185 via a—CH₂—C(═O)—CH₂— linkage. This activated protein was contacted with PL1,the aminated fatty acid derivative containing an aminooxy group whosepreparation is described above, to form an oxime linking the fatty acidderivative to the protein. The conjugate with the attached fatty acidgroup is expected to reduce renal clearance, thus extending thecirculating half-life of the CRM197 protein and increasing itsusefulness as a carrier in conjugate vaccines. Mass spectral data forthe native protein (FIG. 3A, using Method A), the activated protein(FIG. 3B) and the protein conjugate (FIG. 3C) are provided in FIG. 3.

Synthesis of a Site-Defined Azido Compound Bearing CRM197

Method A—

Eluent A: water+0.1% Formic Acid, Eluent B: Acetonitrile++0.1% FormicAcid

Gradient: from 3 to 80% B in 2 min—Flow 1.8 ml/min. Column: AcQuityBEH300 SEC 4.6×30 mm 50° C.

SDS Page Gel Analysis—NuPage 4-12% Bis-Tris Gel; 1.5 mm*10 well

To CRM197 (32.5 mg/ml) (185 μL, 0.103 μmol) in sodium phosphate bufferpH 7.4 (230 μL) was added TCEP HCl (3 mg/mL, water, 58.9 μL, 0.616μmol). The reaction was stirred at room temperature for 16 h followed byaddition of 1,3-dichloropropan-2-one (20 mg/mL, in DMSO, 13.04 μL, 2.054μmol). The reaction was stirred for 3.5 h, then passed through 0.5 mLZeba™ spin size exclusion column (7K MWCO, from Thermo Scientific) andbuffer exchanged to 0.1 M sodium phosphate buffer pH 6 to afford theketone-bearing CRM197 (6.78 mg/ml, 1.3 mL, nanodrop method) LCMS[M+1]=58465

Into a solution of ketone-modified CRM197 (6.78 mg/ml—sodium phosphatebuffer pH6, 1.3 mL, 0.151 μmol) was addedO-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)hydroxylamine (300mg/ml-DMSO) (0.044 mL, 0.045 mmol). The reaction mixture was agitatedfor 36 h at 23° C. then passed through 5 mL Zeba™ spin column elutingwith PBS 7.4 to provide the title compound (4.41 mg/ml, 1.6 mL, 80%yield, Nano drop method)

LCMS [M+1]=58682.5

FIG. 4 shows the SDS Page for the modified CRM197.

Example 2

Starting material preparation: Synthesis ofpE-R—P—R-L-C—H—K-G-P-Nle-C—F—OH (disulfide C⁶-C¹²) (8)

Preparation of Intermediate 8a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-F—OH, Fmoc Removaland Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (40.0 g, 64.0 mmol) was washed with DCM(3×). A solution of Fmoc-F—OH (24.8 g, 64.0 mmol) in DCM (400 mL) andDIPEA (44.7 mL, 256 mmol) was added and the suspension was shaken for 22h at room temperature. The resin was washed thoroughly withDCM/MeOH/DIPEA (17:2:1) (3×), DCM (3×), DMA (3×), DCM (3×). The resinwas then treated four times for 10 min with a mixture of piperidine/DMA(1:4) (400 mL) followed by washing with DMA (2×180 ml). Thepiperidine/DMA solutions and DMA washing solutions were collected fordetermination of the loading of the resin. 1 mL of the combinedsolutions was diluted to 500 mL with MeOH and the UV absorption at 299.8nm was measured to be A=0.368. This corresponds to an Fmoc amount of46.2 mmol. The resin was washed thoroughly with DCM (3×), DMA (3×), DCM(3×) and dried in vacuo to give Intermediate 8a (50.7 g; loading=0.91mmol/g).

Preparation of Intermediate 8b (Assembly of Linear Peptide)

Intermediate 8a (2.64 g, 2.40 mmol) was subjected to solid phase peptidesynthesis on the Prelude™ peptide synthesizer. Coupling was performed asfollows:

Intermediate 8a (2.64 g, 2.40 mmol) was subjected to solid phase peptidesynthesis on the Prelude ™ peptide synthesizer. Coupling was performedas follows: Number of couplings × Synthesis Coupling AA Reaction timecycle  1 C(Trt) 2 × 30 min D  2 Nle 2 × 15 min A  3 P 2 × 15 min A  4 G2 × 30 min A  5 K(Boc) 2 × 15 min A  6 H(Trt) 2 × 15 min A  7 C(Trt) 2 ×60 min D  8 L 2 × 15 min A  9 R(Pbf) 4 × 1 h A 10 P 2 × 15 min A 11R(Pbf) 4 × 1 h A 12 pE 2 × 15 min A

Preparation of Intermediate 8c (Cleavage from the Resin with ProtectingGroup Removal)

Intermediate 8b (2.40 mmol) was carefully washed with DCM (4×). Amixture of 95% aq. TFA/EDT/TIPS (95:2.5:2.5) (50 mL) was added and thesuspension was shaken at room temperature for 1 h. The cleavage solutionwas filtered off, and fresh cleavage solution (35 mL) was added. Thesuspension was shaken at room temperature for 1 h then the cleavagesolution was filtered off. Fresh solution (35 mL) was added and thesuspension was shaken at room temperature for 1 h. The cleavage solutionwas filtered off. The combined cleavage solutions were poured slowlyonto a stirred mixture of cold heptane/diethyl ether (1:1) (500 mL),giving a precipitate. The suspension was stirred at room temperature for2 h and then the precipitate was allowed to settle down. The supernatantwas sucked off with a frit. The residue was washed with coldheptane/diethyl ether (1:1) (2×100 mL), the supernatant was sucked offwith a frit. The solid was dried in high vacuum to afford Intermediate8c as an off-white solid (3.75 g, 1.88 mmol).

Preparation of Cyclic Peptide 8 (Cyclization and Purification)

Intermediate 8c (3.75 g, 1.88 mmol) was dissolved in H₂O (375 mL). Asolution of 50 mM I₂ in AcOH (45.1 mL, 2.26 mmol) was added in oneportion to the stirred solution and the solution was stirred for 10 minat room temperature. 0.5 M Ascorbic acid in H₂O (5.64 mL, 2.82 mmol) wasadded to quench the excess of I₂. The solution was concentrated to neardryness. The reaction was performed in two poroom temperature ions:0.188 mmol scale and 1.69 mmol scale. The crudes were combined forpurification. The crude was purified by preparative HPLC and lyophilizedfrom ACN/H₂O to afford Compound 8 as a white solid (1.53 g, 0.767 mmol).

The pure product was analyzed by analytical HPLC (Analytical method C:t_(R)=3.43 min) and UPLC-MS (Analytical method B; measured:[M+3]/3=512.4; calculated: [M+3]/3=512.6).

This example illustrates formation of an activated protein starting withthe cyclic peptide 8.

Cyclic peptide 8 (12 mg, 6.76 μmol) was dissolved in 50 mM Na phosphatebuffer pH6.5 (1.5 ml), into which was added TCEP HCl (2.91 mg, 10.13μmol) at room temperature. This reaction mixture was stirred for 1 h atroom temperature. Into above solution was added 1,3-dichloropropan-2-one(4.29 mg, 0.034 mmol) at room temperature, which was stirred for 30 minat room temperature. RP-HPLC eluting 15-60% MeCN/water with 0.1% TFAgave activated protein 8d (6 mg, 2.93 μmol, 43.4% yield). HRMS[M+1](method D); 1590.7911 (observed), 1590.7912 (expected).

pE-R—P—R-L-C—H—K-G-P-Nle-C—F—OH with a —S—CH₂—C(═Z)—CH₂—S— linkagebetween the 2 cysteines at position 6 and 12 [C⁶-C¹²], and Z is:

Into a solution of Compound 8((S)-2-((3S,6R,14R,17S,20S,28aS)-17-((1H-imidazol-5-yl)methyl)-20-(4-aminobutyl)-3-butyl-14-((S)-2-((S)-5-guanidino-2-((S)-1-((S)-5-guanidino-2-((S)-5-oxopyrrolidine-2-carboxamido)pentanoyl)pyrrolidine-2-carboxamido)pentanamido)-4-methylpentanamido)-1,4,10,15,18,21,24-heptaoxohexacosahydropyrrolo[2,1-i][1,23,4,7,10,13,16,19]dithiahexaazacyclohexacosine-6-carboxamido)-3-phenylpropanoicacid) (11.5 mg, 5.62 μmol) and(S)-1-(aminooxy)-19-carboxy-2,7,16,21-tetraoxo-9,12-dioxa-3,6,15,20-tetraazaoctatriacontan-38-oicacid compound with 2,2,2-trifluoroacetic acid (1:1) (9.19 mg, 0.011mmol) in 100 nM Na phosphate buffer pH6.0 (1 ml) was added aniline(2.051 μl, 0.022 mmol) at room temperature. Addition of DMSO (50 μl)gave homogeneous solution. This reaction mixture was stirred at roomtemperature for 2 h. RP-HPLC eluting 15-60% MeCN/water with 0.1% TFAgave the expected conjugate,(1-((Z)-((3S,6R,14R,17S,20S,28aS)-17-((1H-imidazol-5-yl)methyl)-20-(4-aminobutyl)-3-butyl-6-((S)-1-carboxy-2-phenylethylcarbamoyl)-14-((S)-2-((S)-5-guanidino-2-((s)-1-((S)-5-guanidino-2-((S)-5-oxopyrrolidine-2-carboxamido)pentanoyl)pyrrolidine-2-carboxamido)pentanamido)-4-methylpentanamido)-1,4,15,18,21,24-hexaoxodocosahydropyrrolo[2,1-i][1,23,4,7,10,13,16,19]dithiahexaazacyclohexacosin-10(1H,9H,11H)-ylidene)aminooxy)-19-carboxy-2,7,16,21-tetraoxo-9,12-dioxa-3,6,15,20-tetraazaoctatriacontan-38-oicacid)

(4.5 mg, 1.646 mol, 29.3% yield). HRMS (method D) [(M+3)/3]; 759.7487(observed), 759.7462 (expected). Retention time 4.12 min.

Example 3

Into a solution of CRM197 (200 μg, 6.2 ul, 0.0034 μmol) in 50 mM Naphosphate buffer pH7.4 (10 μl) was added aqueous solution of TCEP HCl(5.89 μg, 0.021 μmol). This reaction mixture was left for 15 h at roomtemperature. 1,3-dichloropropan-2-one (4.58 μg, 0.034 μmol 10 eq) wasadded into the mixture. This reaction was left at room temperature for 2h. The crude was passed through a Zeba™ size exclusion column. LCMS;[M+1]=58465. This activated protein can be reacted with an aminatedpayload such as a TLR agonist, to form a carrier protein conjugated witha compound that may enhance immune responses to any antigen added to thecarrier protein.

Into a solution of ketone-modified CRM197 (5 mg/ml, Na phosphate buffer,pH6.0) (50 μg, 0.00086 μmol) were addedN-(3-(4-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethyl)piperazin-1-yl)propyl)-2-(aminooxy)acetamide(66.8 μg, 0.064 μmol) and aniline (0.0020 μl, 0.021 μmol). This reactionwas left for 14 h at 23° C. to give the desired conjugate A based onLCMS analysis. Reaction mixture was passed through 0.5 mL Zeba™ sizeexclusion column eluting PBS pH7.2 buffer. LCMS; [M+1]=59032.

Synthesis of PL:

Into a solution of tert-butyl2-(3-bromopropylamino)-2-oxoethoxycarbamate (53.3 mg, 0.171 mmol) and8-methyl-2-(2-methyl-4-(2-(piperazin-1-yl)ethoxy)phenethyl)benzo[f][1,7]naphthyridin-5-amine(52 mg, 0.114 mmol) in DMF (0.5 ml) was added potassium carbonate (39.4mg, 0.285 mmol) at RT, which was stirred for 24 h at RT. water and EtOAcwas added. The organic layer was separated. The aqueous layer wasextracted with EtOAc. The combined organic layer was dried over Na₂SO₄,filtered and concentrated in vacuo to give crude Boc-protected material,this was dissolved in DMF (0.5 ml), into which was added TFA (0.5 mL,6.49 mmol). this was stirred for 30 min at RT. After removal of solvent,RP-HPLC purification eluting 15-60% MeCN/water with 0.1% TFA gaveN-(3-(4-(2-(4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethyl)piperazin-1-yl)propyl)-2-(aminooxy)acetamide(25 mg, 0.024 mmol, 21.02% yield). LCMS; [M+1]=586.

Example 4

The following example uses an anti-VEGF antibody fragment (VEGF-Fab),and a fatty acid derivative that is added to increase serum half-life ofthe antibody fragment. Selective reduction of the inter-chain disulfidein the presence of several less accessible intra-chain disulfidelinkages is achieved using TCEP in PBS at pH 7. The reduced protein isreacted with a dihaloacetone (dibromoacetone or dichloroacetone) toprovide an activated protein having the three-carbon tether—CH₂—C(═O)—CH₂— linking the sulfur atoms together. The activated proteinis contacted with a linker-payload moiety having an aminooxy as thereactive portion to form an oxime with the ketone derived from thedihaloacetone. The linking group L in this example is

where [X] and [PL] indicate the points of attachment for —X—NH₂ andpayload PL, respectively, and the payload is a C18 fatty acid group. Inthe example, X is —ONH₂, which forms an oxime with the carbonyl of theacetonyl ketone of the activated protein. FIG. 5 depicts the formationof the activated protein for this example and shows the mass spectralevidence for its formation.

Into a solution of A (72.72 μg, 6.0 uL, 0.0015 μmol) in PBS pH7.4 (8 μl)was added TCEP HCl (2.63 μg, 0.0092 μmol). This reaction mixture wasleft for 3 h at room temperature. 1,3-dichloropropan-2-one (1.945 μg,0.015 μmol) was added and the reaction was allowed to stand at roomtemperature for 1 h. A was consumed and converted into desired productB. The crude was passed through a size exclusion column. LCMS;[M+1]=47538

Into a solution of B (36.36 μg, 0.00076 μmol) in PBS pH7.4 (22.5 μl)were added (S)-1-(aminooxy)-19-carboxy-2,7,16,21-tetraoxo-9,12-dioxa-3,6,15,20-tetraazaoctatriacontan-38-oicacid compound with 2,2,2-trifluoroacetic acid (1:1) (64.33 μg, 0.079μmol) and aniline (0.00105 μl, 0.011 μmol) at room temperature, whichwas stirred for 14 h at 23° C. B was consumed and converted into desiredproduct C. The crude was passed through a Zeba™ Spin Desalting Column,7K MWCO (from Thermo Scientific)) LCMS; [M+1] (method A)=48222. FIG. 6depicts formation of the protein conjugate and shows mass spectralevidence for conjugate formation.

Example 5

Peptide A (1 mg, 0.519 μmol) was dissolved in buffer (2.5 ml) (50 mMsodium phosphate buffer pH6.5 (1.5 mL), 40% MeCN (0.9 mL), 2.5% DMF (0.1mL)), into which was added TCEP HCl (0.164 mg, 0.571 μmol) at roomtemperature. this reaction mixture was stirred for 60 min.1,3-dibromoacetone (0.164 mg, 0.571 mol) in DMF (0.1 ml) was added intothe reaction mixture at room temperature. After being stirred for 3 min,acetone adduct B was observed to form in quantitative conversion basedon LCMS analysis. LCMS (Method C) [M+2]/2=991.

Example 6: Preparation of antiHer2 Antibody-Drug Conjugates

Method A:

Step 1.

Step 1: Into a solution of Anti-HER2 IgG (20.36 mg/ml in 0.1M Tris/HCl,30 μl, 610.8 μg, 0.0041 μmol) and 1,3-dichloropropan-2-one (66.1 μg,0.495 μmol) was added TCEP HCl (14.17 μg, 0.049 μmol), which wasagitated for 16 h at 4° C. The reaction mixture was passed through 0.5mL Zeba™ spin column eluting PBS buffer (pH7.2). Modification of 4 interchain disulfides was confirmed by analysis with PNGase F (New EnglandBiolab), Endoproteinase Lys-C(Roche) and non reducing/reducing SDS PAGE(4-12% Bis-Tris Gel with colloidal blue staining) performed with samplestaken from the reaction solution. LCMS (method B); 145394 (afterdeglycosylation with PNGase F).

Step 2:

FIG. 7 shows the SDS PAGE for the conjugate and the conjugate followingreduction (see below). SeeBlue Plus2 Pre-Stained Standard (Invitrogen)was used as apparent molecular weights ladder. This demonstrates thatlittle or no unconjugated antibody is present in the conjugationproduct: unconjugated antibody would produce a lower-molecular weightband upon reduction due to dissociation of the antibody held togetheronly by disulfide bonds. The conjugate, having the fragments covalentlylinked through the —S—CH₂—C(═X)—CH₂—S— linkage, cannot dissociate uponreduction.

Method B:

Step 1:

Into a solution of Anti-HER2 IgG (20.36 mg/ml in 0.1M Tris/HCl) (610.8μg, 0.0041 μmol) (30 ul) and 1,3-dichloropropan-2-one (66.1 μg, 0.495μmol) was added TCEP HCl (14.17 μg, 0.049 μmol), which was agitated for16 h at 4° C. The reaction mixture was passed through 0.5 mL Zeba™ spincolumn eluting PBS pH7.2. Successful modification at inter chaindisulfide by 4 acetone formation was confirmed by analysis with PNGase F(New England Biolab), Endoproteinase Lys-C(Roche) and nonreducing/reducing SDS PAGE (4-12% Bis-Tris Gel with colloidal bluestaining) performed with samples taken from the reaction solution. LCMS(method B); 145394 (after deglycosylation). Reduced sample for SDS PAGEwas prepared following the procedure described before.

Step 2:

Into a solution of modified Anti-HER2 IgG (7.14 mg/mL, 100 mM aniliniumacetate buffer pH4.8) (600 μg, 0.0040 μmol) was added(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(aminooxy)-N-methylhexanamido)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid (30 mg/ml, 104 μg, 0.121 μmol) at rt, which was agitated for 19 hat RT. The resulting mixture was passed through 0.5 ml Zeba™ spin columnone time eluting PBS pH7.2. Successful modification of ketones wasconfirmed by analysis with PNGase F (New England Biolab), EndoproteinaseLys-C (Roche) and non reducing/reducing SDS PAGE (4-12% Bis-Tris Gelwith colloidal blue staining) performed with samples taken from thereaction solution. DAR was 3.8. LCMS (method B); 148770 (afterdeglycosylation). SeeBlue Plus2™ Pre-Stained Standard (Invitrogen) wasused as apparent molecular weights ladder. Reduced sample for SDS PAGEwas prepared following the procedure described before. LC-MS data forthe product of Step 2 is shown in FIG. 7.

Example 7: Preparation of Antibody B-DM1 Conjugate

Step 1:

Into a solution of dichloroacetone (7.35 mg, 0.055 mmol, 368 ul) in Trisbuffer (4800 ul) was added antibody B IgG (Antibody B IgG recognizes adifferent antigen from Her2: 68.2 mg, 0.458 μmol, 400 ul), which wascooled to 4° C. for 60 min. TCEP HCl (1.576 mg, 5.50 μmol, 524 ul) at 4°C., which was left for 16 h at 4° C. room. The mixture was concentratedvia 10K Amicon® membrane filtration and diluted with PBS. This cycle wasrepeated by 2 times. After filtration, sample was passed through 5 mlZeba™ desalting column. Successful modification at inter chain disulfideby 4 acetone formation was confirmed by analysis with PNGase F (NewEngland Biolab) and non reducing/reducing SDS PAGE (4-12% Bis-Tris Gelwith colloidal blue staining) performed with samples taken from thereaction solution. LCMS (method B); 146020 (after deglycosylation).Reduced sample for SDS PAGE was prepared following the proceduredescribed before.

Step 2:

PL1 (Method A):

Into a solution of modified Antibody B IgG (48 mg, 0.322 μmol, 1.2 ml)were added DMSO solution of DM-1 derivatives (10.00 mg, 8.05 μmol, 67ul) and 3,5-diaminobenzoic acid (14.70 mg, 0.097 mmol, 30 ul), which wasstirred at 23° C. for 15 h. The mixture was concentrated via 10K Amicon®membrane filtration and diluted with PBS. This cycle was repeated by 3times. After filtration, sample was passed through 5 ml Zeba™ desaltingcolumn. Successful modification of ketones was confirmed by analysiswith PNGase F (New England Biolab). DAR was 4 based on LCMS. LCMS(method B); 150915 (after deglycosylation).

PL1 (Method B):

Into a solution of modified Antibody B IgG (679 μg, 0.0046 μmol) in 0.1MNa phosphate pH6.0 were added2-(aminooxy)-N-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,13-dioxo-6,9,15,18-tetraoxa-3,12-diazaicosan-20-yl)acetamidein DMSO (563 μg, 0.911 μmol, 2.25 ul) at RT, which was stirred for 20 hat 23° C. The reaction mixture was passed through 0.5 ml desaltingcolumn eluting with 100 mM HEPES with EDTA 3 times. Introduction of 3.8maleimide linker/antibody (DAR=3.8) was confirmed by LCMS. LCMS (methodB); 147968 (after deglycosylated with PNGase F (New England Biolab)).

Into a solution of modified Antibody B IgG (177 μg, 0.0012 μmol) in 100mM HEPES buffer with 10 mM EDTA was added DM-1 in DMSO (8.65 μg, 0.012μmol, 0.288 ul) at RT, which was agitated for 6 h at 23° C.N-methylmaleimide (1.3 mg/ml in DMSO) (2.083 μg, 0.019 μmol) was addedinto the reaction solution, which was agitated for 10 min. The reactionmixture was passed through 0.5 mL desalting column eluting 100 mM HEPESbuffer. DAR was 3.7 based on LCMS. LCMS (method B); 153967 (DAR4)(glycosylated).

PL2:

Into a solution of modified Antibody B IgG (420 μg, 0.0028 μmol) inHEPES buffer with 10 mM EDTA was added DM-1 in DMSO (10.61 μg, 0.014μmol, 0.55 ul) at RT, which was agitated for 8 h at RT. The reactionmixture was passed through 0.5 ml desalting column eluting with 100 mMHEPES with EDTA 3 times. The reaction mixture was passed through 0.5 mLdesalting column eluting 100 mM HEPES buffer. DAR was 3.6 based on LCMS.LCMS (method B); 150101 (DAR4) (after deglycosylated with PNGase F (NewEngland Biolab)).

Into a solution of modified Antibody B IgG (420 μg, 0.0028 μmol) inHEPES buffer with 10 mM EDTA was added DM-1 in DMSO (10.61 μg, 0.014μmol, 0.55 ul) at RT, which was agitated for 8 h at RT. The reactionmixture was passed through 0.5 ml desalting column eluting with 100 mMHEPES with EDTA 3 times. The reaction mixture was passed through 0.5 mLdesalting column eluting 100 mM HEPES buffer. DAR was 3.6 based on LCMS.LCMS (method B); 150101 (DAR4) (after deglycosylated with PNGase F (NewEngland Biolab)).

PL3:

Into a solution of modified Antibody B IgG (47.3 mg, 0.317 μmol, 1.1 ml)were added DMSO solution of DM-1 derivatives (6.34 mg, 6.35 mol, 42.3ul) and 3,5-diaminobenzoic acid (13.52 mg, 0.089 mmol, 27 ul), which wasstirred at 23° C. for 15 h. The mixture was concentrated via 10K Amicon®membrane filtration and diluted with PBS. This cycle was repeated by 2times. After filtration, sample was passed through 5 ml Zeba™ desaltingcolumn. Successful modification of ketones was confirmed by analysiswith PNGase F (New England Biolab). DAR was 4 based on LCMS. LCMS(method B); 150910 (after deglycosylation).

PL4:

Into a solution of modified Antibody B IgG (250 μg, 0.0017 μmol, 10 ul)in PBS were added the required alkoxyamine shown above (21.66 μg, 0.025μmol, 0.245 ul) and 3,5-diaminobenzoic acid (383 μg, 2.52 μmol, 0.43 ul)at RT, which was agitated for 24 h at 23° C. The reaction mixture waspassed through 0.5 mL desalting column twice eluting with PBS. DAR was 4based on LCMS. LCMS (method B); 152446 (glycosylated).

Synthesis of PL1, PL2, PL3;

PL1 Synthesis:

tert-butyl(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethyl)carbamate(245 mg, 0.746 mmol) was dissolved in 4N HCl in dioxane (2 mL, 8.00mmol) at RT, which was stirred for 1 h at RT. After removal of solvent,the crude was used for next reaction without further purification.

into a solution of 2-(((tert-butoxycarbonyl)amino)oxy)acetic acid (185mg, 0.970 mmol) and TEA (0.520 mL, 3.73 mmol) in DCM (8 mL) were addedEDC (172 mg, 0.895 mmol) and HOBT (114 mg, 0.746 mmol) at RT, which wasstirred for 5 min at RT.1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-pyrrole-2,5-dione (197 mg, 0.746mmol) in DCM (4 mL) was added into the above reaction mixture. Afterstirring for 1 h, DCM and water were added. The organic layer wasseparated. The aqueous layer was extracted with DCM. The combinedorganic layer was dried over Na₂SO₄, filtered and concentrated in vacuo.RP-HPLC purification eluting 15-65% MeCN/water with 0.1% TFA gavetert-butyl2-((2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethyl)amino)-2-oxoethoxycarbamate(62 mg, 0.154 mmol, 20.70% yield for 2 steps) as a colorless oil. ESI-MS(method A) m/z: 402 [M+1]+, Retention time: 1.60 min. ¹H-NMR (CDCl₃-d,400 MHz); 1.48 (s, 9H), 3.49-3.75 (m, 14H), 6.71 (s, 2H).

Into a solution of tert-butyl2-((2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethyl)amino)-2-oxoethoxycarbamate(62 mg, 0.154 mmol) in DCM (400 μl) was added TFA (400 μl) at RT, whichwas stirred for 1 h at RT. After removal of solvent, the crude was putin vacuum for O.N. used without further purification. ESI-MS (method A)m/z: 302[M+1]⁺

PL2 Synthesis:

Into a suspension of 2-Cl Trt resin (1.70 mmol/g) (0.086 g, 1.7 mmol)and 1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecan-12-oic acid (2g, 5.19 mmol) in DCM (8 mL)/DMF (4 mL) was added DIPEA (2.67 mL, 15.30mmol) dropwise, which was stirred for 15 h at RT. Solvent was drained.The resin was rinsed with DCM/MeOH/DIPEA (17/2/1, 40 ml), DCM (8 mL*2),DMF (8 mL*2), DCM (8 mL*2) and dried in vacuo.

Resin (0.679 g, 1.7 mmol) was charged into reaction vessel. 5 mL of 20%Piperidine in DMF was added, which was stirred gently for 1 min andremoved. Another 10 mL of 20% Piperidine in DMF was added, waited for 20min with intermittent stirring and removed. DMF (10 ml) was addedstirred for 15 s and removed via vacuum filtration. Repeated this stepfour times (check with Kaiser test; positive, violet-deep blue).Solution of HOAt (0.463 g, 3.40 mmol) and2-(((tert-butoxycarbonyl)amino)oxy)acetic acid (0.650 g, 3.40 mmol) inDMF (8 mL) was added into resin and DIC (0.530 mL, 3.40 mmol) in DMF (4mL) was added. reaction mixture was agitated for 1.5 h at RT. Resin wasfiltered off, rinsed with DMF (10 ml) four times and dried in vacuo.

Resin (0.926 g, 1.7 mmol) was charged into reaction vessel. 5 mL of 20%Piperidine in DMF was added, which was stirred gently for 1 min andremoved. Another 10 mL of 20% Piperidine in DMF was added, waited for 20min with intermittent stirring and removed. DMF (10 ml) was added,stirred for 15 s and removed via vacuum filtration. Repeated this stepfour times (check with Kaiser test; positive, violet-deep blue).Solution of HOAt (0.463 g, 3.40 mmol) and2-(((tert-butoxycarbonyl)amino)oxy)acetic acid (0.650 g, 3.40 mmol) inDMF (8 mL) was added into resin and DIC (0.530 mL, 3.40 mmol) in DMF (4mL) were added. The reaction mixture was agitated for 1.5 h at RT. Resinwas filtered off, rinsed with DMF (10 ml) four times and dried in vacuo.

Resin was suspended with 30% HFIP (hexafluoroisopropanol) in CHCl₃ (20mL, 1.700 mmol), which was agitated for 2 h at RT. Solvent drained wasconcentrated to give crude2,2-dimethyl-4,8,17-trioxo-3,6,1,15,71,74-hexaoxoxa-5,9,18-triazahexacosan-26-oicacid (1.13 g, 2.347 mmol, 138% yield). This was used for the nextreaction without further purification. ESI-MS m/z: 482[M+1]+, Retentiontime: 1.10 min (method B).

Into a solution of2,2-dimethyl-4,8,17-trioxo-3,6,12,15,21,24-hexaoxa-5,9,18-triazahexacosan-26-oicacid (819 mg, 1.7 mmol) in DMF (6 mL) were added HOAt (463 mg, 3.40mmol) and DIC (0.530 mL, 3.40 mmol) at RT respectively, which wasstirred for 5 min at RT. Into above mixture were added1-(2-aminoethyl)-1H-pyrrole-2,5-dione (518 mg, 2.040 mmol) and DIPEA(diisopropyl ethylamine, 0.594 mL, 3.40 mmol) at RT, which was stirredfor 1 h at RT. The reaction mixture was diluted with water and EtOAc(ethyl acetate). The organic layer was separated. The aqueous layer wasextracted with EtOAc. The combined organic layer was dried over Na₂SO₄,filtered and concentrated in vacuo. The desired compound was mostly inaqueous layer based on LCMS. After lyophilization of aqueous layer, Thecrude was purified via RP-HPLC eluting 15-70% MeCN/water with 0.1% TFAgave tert-butyl(23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,11,20-trioxo-6,9,15,18-tetraoxa-3,12,21-triazatricosyl)oxycarbamate(600 mg, 0.994 mmol, 58.5% yield). ESI-MS m/z: 604[M+1]+, Retentiontime: 1.14 min (method B). ¹H-NMR (CDCl3-d, 400 MHz); 1.48 (s, 7.5H),1.55 (s, 1.5H), 3.47-3.71 (m, 20H), 3.97 (s, 2H), 4.04 (s, 2H), 4.37 (s,1.65H), 4.47 (s, 0.35H), 6.72 (s, 2H).

Into a solution of tert-butyl(23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,11,20-trioxo-6,9,15,18-tetraoxa-3,12,21-triazatricosyl)oxycarbamate(8.4 mg, 0.014 mmol) in DCM (100 μl) was added TFA (100 μl) at RT, whichwas agitated for 1 h at RT. Removal of solvent resulted in2-(aminooxy)-N-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,13-dioxo-6,9,15,18-tetraoxa-3,12-diazaicosan-20-yl)acetamide.This was used for next reaction without further purification. ESI-MSm/z: 504[M+1]+, Retention time: 0.69 min (method A).

PL3 Synthesis:

Into a solution of2-(aminooxy)-N-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,13-dioxo-6,9,15,18-tetraoxa-3,12-diazaicosan-20-yl)acetamide(22.79 mg, 0.031 mmol) in DMA (0.6 mL) was added DM-1 (23 mg, 0.031mmol) and 100 mM Na phosphate pH7.4 (0.600 mL) at 5° C. DIPEA (10.88 μl,0.062 mmol) was added at the same temperature. This reaction mixture wasletting warm to RT and stirred for 1.5 h. The reaction mixture wasdiluted with DCM and sat. sodium bicarbonate aq. The organic layer waswashed with sat. NH₄Cl (aq) and brine. The combined organic layer wasdried over Na₂SO₄, filtered and concentrated in vacuo. Silica gelchromatography eluting with 0-15% MeOH/DCM gave the desired compound (21mg, 0.017 mmol, 54.3% yield). ESI-MS m/z: 1242[M+1]+, Retention time:1.00 min (method A). ¹H-NMR (CDCl3-d, 400 MHz); 0.80 (s, 3H), 1.21-1.33(m, 9H), 1.41-1.51 (m, 1H), 1.56-1.59 (m, 1H), 2.31-2.39 (m, 1H),2.57-2.65 (m, 2H), 2.79-2.88 (m, 1H), 2.86 (s, 3H), 2.91-3.13 (m, 5H),3.16-3.24 (m, 1H), 3.20 (s, 3H), 3.36 (s, 3H), 3.43-3.76 (m, 25H), 3.90(d, J=3.6 Hz, 2H), 3.98 (s, 3H), 4.02 (s, 2H), 4.17 (s, 2H), 4.25-4.32(m, 1H), 4.77-4.80 (m, 1H), 5.30-5.37 (m, 1H), 5.62-5.69 (m, 1H), 6.26(s, 1H), 6.38-6.45 (m, 1H), 6.63-6.68 (m, 2H), 6.82-6.84 (m, 1H), 6.92(brs, 1H), 7.14-7.24 (2H).

Into a solution of tert-butyl(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (320 mg, 1.289 mmol) in DCM(3 mL) were added 2-bromoacetyl bromide (0.225 mL, 2.58 mmol) and DIPEA(0.563 mL, 3.22 mmol) at 5° C., which was stirred for 15 min lettingwarm to RT. After removal of solvent, silica gel column chromatographypurification eluting 0-40-100% EtOAc/heptane gave tert-butyl(2-(2-(2-(2-bromoacetamido)ethoxy)ethoxy)ethyl)carbamate (317 mg, 0.858mmol, 66.6% yield). ESI-MS m/z: 269[M+1-Boc]+, Retention time: 1.41 min(method A). H-NMR (CDCl3, 400 MHz); 1.45 (s, 9H), 3.34 (brs, 2H,3.48-3.52 (m, 2H), 3.53-3.60 (m, 4H), 3.63 (s, 4H), 3.88 (s, 2H).

Into a solution of tert-butyl(2-(2-(2-(2-bromoacetamido)ethoxy)ethoxy)ethyl)carbamate (317 mg, 0.858mmol) in DCM (1 mL) was added TFA (1 mL), which was stirred for 30 minat RT. After removal of solvents, the resulting crude was used for nextreaction without further purification.

Into a solution of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-2-bromoacetamide(329 mg, 0.858 mmol) in DCM (1.5 mL) were added pre-activated ester(prepared from 2-(((tert-butoxycarbonyl)amino)oxy)acetic acid (328 mg,1.716 mmol), HOAt (175 mg, 1.287 mmol) and DIC (0.267 mL, 1.716 mmol) inDMF (1.5 mL) being stirred for 5 min at RT) and DIPEA (0.749 mL, 4.29mmol) at 5° C., which was stirred for 20 min letting warm to RT. EtOAcand water was added. The organic layer was separated. the aqueous layerwas extracted with EtOAc. The combined organic layer was dried overNa2SO4, filtered and concentrated in vacuo. Silicagel chromatographypurification eluting 0-5% MeOH/DCM tert-butyl(14-bromo-2,13-dioxo-6,9-dioxa-3,12-diazatetradecyl)oxycarbamate (150mg, 0.339 mmol, 39.5% yield). ESI-MS m/z: 343[M+1-Boc]+, Retention time:1.30 min ((method A). H-NMR (CDCl₃, 400 MHz); 1.49 (s, 9H), 3.47-3.55(m, 4H), 3.59-3.63 (m, 4H), 3.65 (s, 4H), 3.88 (s, 2H), 4.34 (s, 2H).

tert-butyl(14-bromo-2,13-dioxo-6,9-dioxa-3,12-diazatetradecyl)oxycarbamate (150mg, 0.339 mmol) was dissolved in DCM (Volume: 1 mL, Ratio: 1.000), intowhich was added TFA (Volume: 1, Ratio: 1.000) at RT. this reactionmixture was stirred for 30 min at RT. After removal of solvent, RP-HPLCeluting with 10-25% MeCN/water containing 0.1% TFA gave2-(aminooxy)-N-(2-(2-(2-(2-bromoacetamido)ethoxy)ethoxy)ethyl)acetamide(110 mg, 0.241 mmol, 71.1% yield). ESI-MS m/z: 344[M+2]+, Retentiontime: 0.44 min (method A).

Into a solution of2-(aminooxy)-N-(2-(2-(2-(2-bromoacetamido)ethoxy)ethoxy)ethyl)acetamide(42.9 mg, 0.075 mmol) in DMA (1 mL) was added DM-1 (37 mg, 0.050 mmol)and 75 mM Na phosphate pH8.5 (1 mL) at 5° C. DIPEA (0.026 mL, 0.150mmol) was added at the same temperature. This reaction mixture wasletting warm to RT and stirred for 1 h. reaction mixture was dilutedwith DCM and sat. sodium bicarbonate aq., and the organic layer waswashed with sat. NH₄Claq and brine. combined organic layer was driedover Na₂SO₄, filtered and concentrated in vacuo. Silica gelchromatography eluting with 0-15% MeOH/DCM gave the desired compound (31mg, 0.031 mmol, 61.9% yield). ESI-MS m/z: 1000[M+1]+, Retention time:1.61 min (method A). ¹H-NMR (CDCl3-d, 400 MHz); 0.79 (s, 3H), 1.20-1.33(m, 9H), 1.41-1.50 (m, 2H), 2.17-2.22 (m, 1H), 2.50-2.63 (m, 2H),2.70-2.81 (m, 2H), 2.86-2.94 (m, 3H), 2.99-3.01 (m, 1H), 3.10-3.13 (m,1H), 3.18-3.20 (m, 4H), 3.36 (s, 3H), 3.45-3.62 (m, 14H), 3.98 (s, 3H),4.17 (s, 2H), 4.25-4.31 (m, 1H), 4.78-4.82 (m, 1H), 5.30-5.35 (m, 1H),5.63-5.69 (m, 1H), 6.27 (s, 1H), 6.39-6.45 (m, 1H), 6.61-6.64 (m, 2H),6.83 (s, 1H), 6.87 (brs, 1H).

Example 8: Antibody C Fab Conjugate

Step 1:

Into a solution of Antibody C Fab (Antibody C binds a different targetantigen from Her2 and Antibody B: 1668 μg, 0.035 μmol, 120 ul) in 100 mMNa phosphate with EDTA, pH7.4 was added TCEP HCl (35.2 μg, 0.123 μmol,11.73 ul) at RT, which was agitated for 1.5 h at 23° C.1,3-dichloropropan-2-one (117 μg, 0.878 μmol, 5.85 ul) was added intothe reaction mixture, which was agitated for 40 min at 23° C. 1 acetonebridge modification was observed by LCMS. The reaction mixture waspassed through 0.5 desalting column eluting with 100 mM NaOAc bufferpH5.2. LCMS (method B); 47554.

Step 2:

Into a solution of modified Antibody C Fab (1668 μg, 0.035 μmol, 148 ul)in 100 mM NaOAc buffer pH5.2 and the aminooxy-substituted fatty acidshown (1852 μg, 2.63 μmol) was added 3,5-diaminobenzoic acid (694 μg,4.56 μmol, 5.34 ul) at RT, which was agitated for 20 h at 23° C.Additional aminooxy-fatty acid (1852 μg, 2.63 μmol) was added into themixture, which was agitated for 24 h at RT. The reaction mixture waspassed through 5 ml desalting column eluting with PBS pH7.4 to give theexpected Antibody C Fab-fatty acid conjugate (30% yield). LCMS (methodB); 48238.

SDS PAGE image and mass spectrum for the conjugate are provided in FIG.9.

The invention claimed is:
 1. A method to form a Protein-Payloadconjugate from a protein that comprises at least two cysteine residues,where the method comprises: a) contacting the protein with afunctionalized tethering compound under conditions where thefunctionalized tethering compound reacts with two free thiol groups onthe protein to form an activated protein having the two thiol groupscovalently linked together by a functionalized tether, wherein thefunctionalized tethering compound is a dihalo acetone derivative, andthe functionalized tether is —CHR—C(═Z)—CHR—, where Z is O or NR′, andeach R is independently H, phenyl, C1-C4 alkoxy, or C1-C4 alkyl, and R′represents a linking group attached to a payload; and b) if thefunctionalized tether is not already linked to a payload, contacting theactivated protein with a functionalized payload compound to form acovalent attachment of the payload with the functionalized tether toform a Protein-Payload conjugate, wherein the functionalized payload isa compound of the formula H₂N—X-L-PL, where X is NH, N(C1-C4 alkyl),N-L²-PL², or O, each of L and L² independently represents a linkinggroup; and each of PL and PL² independently represents at least onePayload group.
 2. The method of claim 1, wherein the Protein has twocysteine residues joined together by a disulfide linkage before it iscontacted with the functionalized tethering compound, and wherein thedisulfide is reduced to provide a protein with two free cysteineresidues for use in step a) of claim
 1. 3. The method of claim 1,wherein the functionalized payload is a compound of the formulaH₂N—O-L-PL.
 4. The method of claim 1, wherein the activated Proteincomprises a ketone having the formula

where the circle represents the protein, and each sulfur atom is thesulfhydryl of a cysteine residue of the protein.
 5. The method of claim1, wherein the Protein-Payload conjugate comprises a group of theformula:

wherein X is O or NH, L represents a Linker, and PL represents at leastone Payload group.
 6. The method of claim 1, wherein the Protein is anantibody or a vaccine carrier.
 7. The method of claim 1, wherein thePayload comprises a therapeutic agent, a detectable label or a bindinggroup.
 8. The method of claim 6, wherein the Payload comprises anantigen.
 9. The method of claim 6, wherein L comprises a cleavablelinking moiety.
 10. The method of claim 1, wherein L comprises at leastone amino acid.
 11. The method of claim 1, wherein L comprises at leastone spacer selected from: (a) a bond, —O—, —S—, —NH—,—N((C₁-C₆)alkyl)H—, —NH—C(O)—NH—, —C(O)—NH—, —NH—C(O)—; (b)(C₁-C₂₀)alkylene, (C₂-C₂₀)alkenylene, (C₂-C₂₀)alkynylene,—Z—(C₁-C₂₀)alkylene-, —Z—(C₂-C₂₀)alkenylene, —Z—(C₂-C₂₀)alkynylene,(C₁-C₂₀)alkylene-Z—(C₁-C₂₀)alkylene,(C₂-C₂₀)alkenylene-Z—(C₂-C₂₀)alkenylene,(C₂-C₂₀)alkynylene-Z—(C₂-C₂₀)alkynylene, where Z is —NH—,—N(C₁-C₆)alkyl)H—, —NH—C(O)—NH—, —C(O)—NH—, —NH—C(O)—,(C₃-C₇)cycloalkylene, phenylene, heteroarylene, or heterocyclene andwhere said (C₁-C₂₀)alkylene, said (C₂-C₂₀)alkenylene, and said(C₂-C₂₀)alkynylene moieties each independently optionally contain 1-10oxygen atoms interspersed within said moieties; (c)(C₃-C₇)cycloalkylene, (C₃-C₇)cycloalkylene-Y—(C₃-C₇)cycloalkylene,—Y—(C₃-C₇)cycloalkylene, phenylene, —Y-phenylene, phenylene-Y-phenylene,heteroarylene, Y-heteroarylene, heteroarylene-Y-heteroarylene,heterocyclene, —Y-heterocyclene, or heterocyclene-Y-heterocyclene, whereY is (C₁-C₂₀)alkylene, (C₂-C₂₀)alkenylene, (C₂-C₂₀)alkynylene, —O—,—C(O)—, —S—, —NH—, —N((C₁-C₆)alkyl)H—, —NH—C(O)—NH—, —C(O)—NH—, or—NH—C(O)— and where said (C₃-C₇)cycloalkylene, said phenylene, saidheteroarylene, and said heterocyclene moieties are each individuallyoptionally substituted with 1 to 3 substituents selected from halo,(C₁-C₄)alkyl or halo-substituted(C₁-C₄)alkyl; (d) —[OCH₂CH₂]_(v)—, wherev is 1-2,000; and (e) a peptide comprising 1 to 100 amino acids.
 12. Themethod of claim 1, wherein the Protein-Payload conjugate is of theformula:

wherein the circle represents a protein having at least 5 amino acids; Xis NH, N(C₁₋₄ alkyl), N-L²-PL², or O; L and L² are each a linking groupand can be the same or different; and PL and PL² are Payload groups thatcan be the same or different.
 13. The method of claim 12, wherein X isO.
 14. The method of claim 12, wherein the protein is an antibody or avaccine carrier.
 15. The method of claim 12, wherein the payload is atherapeutic agent, a detectable label, an antigen, or a binding group.16. The method of claim 12, wherein L comprises a cleavable linker. 17.The method of claim 12, wherein L comprises at least one amino acid. 18.The method of claim 12, wherein L comprises at least one spacer of theformula (CH₂)₁₋₆ or (CH₂CH₂O)₁₋₄.
 19. The method of claim 1, wherein L²comprises a cleavable linking moiety.
 20. The method of claim 1, whereinL² comprises at least one amino acid.
 21. The method of claim 1, whereinL² comprises at least one spacer selected from: (a) a bond, —O—, —S—,—NH—, —N((C1-C₆)alkyl)H—, —NH—C(O)—NH—, —C(O)—NH—, —NH—C(O)—; (b)(C₁-C₂₀)alkylene, (C₂-C₂₀)alkenylene, (C₂-C₂₀)alkynylene,—Z—(C₁-C₂₀)alkylene-, —Z—(C₂-C₂₀)alkenylene, —Z—(C₂-C₂₀)alkynylene,(C₁-C₂₀)alkylene-Z—(C₁-C₂₀)alkylene,(C₂-C₂₀)alkenylene-Z—(C₂-C₂₀)alkenylene,(C₂-C₂₀)alkynylene-Z—(C₂-C₂₀)alkynylene, where Z is —NH—,—N(C₁-C₆)alkyl)H—, —NH—C(O)—NH—, —C(O)—NH—, —NH—C(O)—,(C₃-C₇)cycloalkylene, phenylene, heteroarylene, or heterocyclene andwhere said (C₁-C₂₀)alkylene, said (C₂-C₂₀)alkenylene, and said(C₂-C₂₀)alkynylene moieties each independently optionally contain 1-10oxygen atoms interspersed within said moieties; (c)(C₃-C₇)cycloalkylene, (C₃-C₇)cycloalkylene-Y—(C₃-C₇)cycloalkylene,—Y—(C₃-C₇)cycloalkylene, phenylene, —Y-phenylene, phenylene-Y-phenylene,heteroarylene, Y-heteroarylene, heteroarylene-Y-heteroarylene,heterocyclene, —Y-heterocyclene, or heterocyclene-Y-heterocyclene, whereY is (C₁-C₂₀)alkylene, (C₂-C₂₀)alkenylene, (C₂-C₂₀)alkynylene, —O—,—C(O)—, —S—, —NH—, —N((C₁-C₆)alkyl)H—, —NH—C(O)—NH—, —C(O)—NH—, or—NH—C(O)— and where said (C₃-C₇)cycloalkylene, said phenylene, saidheteroarylene, and said heterocyclene moieties are each individuallyoptionally substituted with 1 to 3 substituents selected from halo,(C₁-C₄)alkyl or halo-substituted(C₁-C₄)alkyl; (d) —[OCH₂CH₂]_(v)—, wherev is 1-2,000; and (e) a peptide comprising 1 to 100 amino acids.